Optimization of TNF-alpha, overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies

Abstract

Tumor necrosis factor-alpha (TNF-alpha) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-alpha is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-alpha inhibitors. In this work, induction conditions for the production of human TNF-alpha fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD600nm), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-alpha. An 11% increase of TNF-alpha production was achieved after determination of the optimum induction conditions: OD600nm prior induction 0.55, a post induction temperature of 25 degrees C, an IPTG concentration of 1 mM and a post-induction time of 4 h. We have also studied TNF-alpha oligomerization, the major property of this protein, and a K-d value of 0.26 nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable Kd value for protein trimerization probably due to the complexibility of our model. (C) 2012 Elsevier Inc. All rights reserved.